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Vwol 64, 1271-1277
69. Hung, S -L , Srimvasan, S , Frtedman, H M , Etsenberg, R. J , and Cohen, G H
(1992) Structural basis of C3b bmdmg by glycoprotem C of herpes simplex vnus
J Vwol 66,4013%4027
70 (ayan, A M , Dolter, K E , Langeland, N., Gouts, W F., Glortoso, J C , Haarr,
L., and Crumpacker, C S. (1993) Resistance of herpes simplex vn-us type 2 to
neomycin maps to the N-termmal portton ofglycoprotem C J Vwol 67,2434-244 1
71 Haarr, L , Marsden, H S , Preston, C M , Smtley, J R., Summers, W. C., and
Summers, W P (1985) Utillzatton of mternal AUG codons for mttatton of pro-
tem synthesis directed by mRNAs from normal and mutant genes encoding her-
pes simplex virus-specified thymtdme kmase J Vzrol 56,5 12-5 19
72. Marsden, H S., Haarr, L., and Preston, C. M. (1983) Processmg of herpes stm-
plex proteins and evidence that translation of thymtdme kmase mRNA IS mttlated
at three separate AUG codons J Vu-01 46,434445
73. Parrts, D. S., Cross, A, Haarr, L , Orr, A, Frame, M C, Murphy, M , McGeoch,
D J , and Marsden, H S (1988) Identlficatton of the gene encoding the 65-krlo-
dalton DNA-binding protein of herpes simplex virus type- 1 J Vu-ol 62,8 18-825
74. MacLean, G , Rtxon, F , Langeland, N , Haarr, L., and Marsden, H. S (1990)
Identtficatton and charactertsation of the virlon protein products of herpes stm-
plex type-l gene UL47 J Gen Vwol 71,2953-2960
75. Stsk, W. P., Bradley, J. D., Leopold, R J., Stoltzfus, A M , de Leon, M P., Half,
M , Peng, C , Cohen, G H , and Etsenberg, R J. (1994) High level expression and
purtficatlon of secreted forms of herpes simplex virus type-l glycoprotem gD
synthestsed by baculovnus-infected insect cells J Vu-01 68,766-775
76 Harris, M. and Coates, K (1993) Identrficatton of cellular proteins that bmd to the
human tmmunodeficiency virus type-l nef gene product zn vztro a role for
myrtstylation. J Gen Vwol 74, 1581-1589
77 Vandekerckhove, J., Bauw, G , Puype, M , Dan-me, J V , and Montagu, M V
(1985) Protem-blotting on polybrene-coated glass-fiber sheets A basis for acrd
hydrolysis and gas-phase sequencmg of ptcomole quantmes of protein previously
separated on sodium dodecyl sulphatelpolyacrylam˜de gel Eur J Blochem
152,9-l 9.
78 Aebersold, R. H., Teplow, D B , Hood, L E , and Kent, S. B. H (1986)
Electroblottmg onto activated glass. High efficiency preparation of proteins from
119
Analysis of HSV Poiypeptldes
analytical sodium dodecyl sulphate-polyacyrlamide gels for direct sequence analy-
sis. J Bzol Chem 261,4229-4238.
79 Patterson, R. M , Whttcher, L L , He, C , Selkirk, J K , and Merrick, B A. (1993)
Improved protein detectron with a polyvinyhdene fluoride transfer membrane for
two-dimensional gel electrophoresis Blo Techniques 14,752,753
80 Davison, M. D., Rixon, F J , and Davison, A. J (1992) Identificatron of genes
encoding two capsld proteins (VP24 and VP26) of herpes simplex vn-us type- 1 J
Gen Virol 73,2709-2713.
81 Booy, F. P., Newcomb, W. W., Trus, B. L., Brown, J. C., Baker, T. S., and Steven,
A C. (199 1) Liquid-crystalline, phage-like packing of encapsldated DNA m her-
pes simplex vnus Cell 64, 1007-l 0 15
82 Gibson, W and Rolzman, B (1972) Proteins specified by herpes simplex virus
VIII. Characterisatlon and compostion of multiple capsld forms of sub-types 1
and 2. J Vu-01 10, 1044-1052
83. Szilagyr, J F. and Cunningham, C (1991) Identification and characterization of a
novel non-infectious herpes simplex virus-related particle. J Gen Vzrol 72,
661-668.
84. Braun, D K., Rolzman, B., and Pereira, L (1984) Characterrzatron of post-trans-
lational products of herpes simplex vnus gene 35 proteins bmdmg to the surfaces
of full capsids but not empty capsides. J Vzrof 49, 142-153
85. Markwell, A K and Fox, C F. (1978) Surface-specific lodmatlon of membrane
proteins ofviruses and eukaryottc cells using 1,3,4,6-tetrachloro-3a, Ba-dlphenyl-
glucoluril. Biochemzstry 17,4807-4817.
86 Skulstad, S., Rodahl, E , Jakobsen, K., Langeland, N , and Haarr, L (1995) Label-
ing of surface proteins of herpes simplex vnus type 1 using a modified blotm-
streptavidm system. Vzrus Res 37,253-270.
87. Hope, R. G., Palfreyman, J , Suh, M , and Marsden, H. (1982) Sulphated glyco-
proteins induced by herpes simplex vtrus. J Gen Vzrol 58, 399-4 15.
88. Preston, C. M and McGeoch, D (1981) Identification and mapping of two
polypeptides encoded wrthm the herpes simplex vnus type 1 thymrdme kmase
gene sequences. J Vu-01 38,593-605
8
Protein Purification
Joseph Conner


1. Introduction
The isolatton of an indivrdual polypepttde from a heterogeneous mix is an
essential process m characterizing a protein of interest. In purified form a pro-
tein can be used to generate specific polyclonal and monoclonal antibodies for
in vivo studies, in vitro the enzymic properties or interactions with nucleic
acids or other protems can be studied in detail and related to m viva function
and, ultimately, the purified protem can be used in structural determinations
that define how polypepttde chains fold and ammo acids interact to create a
protein with a specttic function. Protein purificatton exploits the properttes a
polypeptide derives from rts unique ammo acid composition and separation
techniques rely on variations rn solubility, size, charge, hydrophobicity and
specific affinities to achieve fracttonation. A combmation of these methods IS
suftictent to isolate an mdivtdual protein from a complex mix. A prerequisite
for any purification is the ability to unambiguously distmguish the protein of
mterest at all stages.This can be achieved by sodium dodecyl sulfate polyacry-
lamide gel electrophoresis (SDS-PAGE), Western blottmg with specific antis-
era, or by use of an assay specific for an activity of the protein.
An enormous range of reagents 1savailable to assist m protein fractionation
and these include compounds such as ammonium sulfate or polyethylene
glycol, which promote protein precipitation, and numerous matrices for size
fractionatton, ion-exchange, hydrophobicity interaction, and affimty chromato-
graphies. These latter techniques are generally performed in columns m which
the matrix forms a stationary phase through which the heterogeneous protein
mixture in an appropriate buffer (the hquid phase) is passed. Proteins that
interact with the stationary phase and are nnmobil˜zed on the column can be
eluted by alterations, such as iomc strength or pH, in the hquid phase. Numer-
From Methods m Molecular Medicme, Vol 10 Herpes Stmplex Virus Profocols
Ed&d by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

121
Conner
122

ous automated chromatography systems,such as FPLC or HPLC, are available
to perform column chromatography although similar results can be achieved
using a peristaltic pump and gradient mixer Ion-exchange and affinity chro-
matographies also can be performed m simple batch procedures.
Size fractionation by gel filtration uses a matrix, comprised of gel par-
ticles in bead form, with a defined pore size. The passage of a protein through
the stationary phase of the column is dependent on its physical size. Small
molecules permeate the matrix, taking longer to elute from the column than
larger molecules that are excluded from the gel bed and move through the
liquid phase out with the matrix. The molecular size separation range of a gel
filtration column depends on the pore size of the gel beads and a variety of
matrices with different separation ranges IS available.
Ion-exchange chromatography is a very versatile technique that utilizes
either positively or negatively charged matrices to adsorb proteins m low
ionic strength buffers. The net charge of a protein is influenced by the buffer
pH and this affects its ability to interact with an ion-exchange matrix. The
isoelectric point (pl) of a protein ts the pH at which it has no net charge. By
varying the pH above or below the pl, the net negative or positive charge of
a protein will be affected and this influences the strength of the interaction
with anionic or catiomc exchange matrices. Protems that bmd to ion exchange
columns are eluted by gradients of salts, usually NaCl or KCl, which com-
pete for charged residues; the stronger the mteraction with the matrix, the
higher the concentration of salt required for elution
Hydrophobicity interaction chromatography (HIC) uses matrices with
hydrophobic groups that interact with surface hydrophobic regions of pro-
teins. High salt concentrations promote and stabilize hydrophobic mterac-
tions and proteins are adsorbed onto HIC columns at high iomc strength.
Elution from the matrix is achieved by reducing the ionic strength of the
buffer.
Affinity chromatography encompasses a wide diversity of matrices that
exploit specific mteractions that proteins may possess. The affinity may be
derived from an antibody, specific or nonspecific nucleic acid sequences,
proteins, or pepttdes with which the protein to be purified is known to inter-
act, substrates or cofactors to which the protein binds or more general matri-
ces with immobilized hgands such as heparm, hydroxylapatite, or Cibacron
blue. Proteins are absorbed onto the matrtx at low ionic strength and eluted
by competition with a relevant ligand or an increase m ionic strength. The
advantage of affinity chromatography is the specificity of the protem for the
immobilized hgand, although, m some instances, such as antibody affinity
chromatography, the strength of the interaction may be so great that condi-
tions required for elution may be detrimental to the protein.
123
Protein Punfica tion

Recent advances m cloning techniques have allowed the expression of pro-
teins wtth addtttonal ammo acid sequences at the N- or C-terminus that con-
fer specific affinities (e.g., glutathione S-transferase-glutathione, histtdine
hexamer-Ni2+) to the protein and assist m purtfication. Plasmids and other
reagents required for producing tagged proteins are available from various
companies that provtde reagents for molecular biology and thts methodology
represents a significant advance in protein purification techniques.
It is beyond the scope of a single chapter to consider all aspects of protein
purification but, by describing the purification of two proteins from herpes
simplex virus (HSV), some insight mto protein fractlonatton techniques will
be gained. The proteins Involved are the Rl and R2 subunits of HSV ribo-
nucleotide reductase, an essential enzyme for viral DNA synthesis and po-
tential target for antiviral chemotherapy (2). Rl and R2 interact to produce
the active form of the enzyme and a peptide, corresponding to the nine ammo
acids at the C-terminus of the R2 subunit, specrfically inhibits enzyme activ-
ity by preventing subunit interaction (2). Structural studies on the protem-
peptrde mteractlon will be of benefit in destgning peptrdomtmetic compounds
as antiviral reagents The proteins were required m large amounts for struc-
tural studies and were overexpressed in Escherlchia colz using the T7 ex-
pression system (3,4); the construction of the expression plasmtds IS
described in detail m (5,6). The purification of R2 demonstrates the effec-
tiveness of ion exchange chromatography in generating large amounts of
homogeneous preparations whereas RI purtfication was comphcated by a
number of factors that prevented the use of this technique and a method for
screening a large number of matrtces, to identify those that provide useful
purtficatton steps, IS described.

2. Materials
1. Equipment: A Pharmacla (Uppsala, Sweden) FPLC system with conductivity
motutor should be used for all column chromatography steps. Chromatofocusing
and anion exchange chromatography are performed on Pharmacia Mono P and
Mono Q columns respectively Frve-milliliter Econo-Pat Heparm Aftigel and
Affigel Blue columns are obtained from Bra-Rad (Hercules, CA). For buffer
exchange, a Pharmacra Fast Desalt column was used. Sodium-dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and Western blottmg are per-
formed with Protean II mnn-gel and Trans-blot kits (Blo-Rad).
2. HEPES buffer: 25 mA4 HEPES, pH 7.6, with 2 n&I dnhrothreltol. Lysozyme IS
obtained from Sigma
3. (NH&SO4 was used erther as a saturated solution or as a finely ground powder
4. Polybuffer 74 (Pharmacra) and the pH of this and the trrethanolamme buffer
should be adjusted with a saturated solution of immodracetrc acid. These buffers
are recommended by Pharmacta for use with the Mono P column.
Conner
124

5 Bu-Tris should be obtained from Sigma and the pH adjusted with concentrated
HCl. Other biologtcal buffers also are obtained from Sigma and used as recom-
mended by Pharmacta m mstructtons provided with Mono Q and Mono S columns
6. Ctbacron blue, reactive red, reacttve yellow and ATP agaroses should be obtamed
from Sigma. Heparin affigel and hydroxylapatite should be obtained from Bto-
Rad and phosphocellulose, from Whatman (Matdstone, UK)
7 CNBr-activated Sepharose should be obtained from Pharmacta and protem cou-
pling performed as recommended m the manufacturer™s mstructtons The
nonapepttde YAGAVVNDL IS synthesized by contmuous flow F-mot chemtstry
(7,s) on a Novabtochem peptide synthestzer

3. Methods
1, E co11 expressing Rl and R2 are lysed by the additton of lysozyme to produce an
mrtlal crude extract Cells from 1 L of culture are harvested by centrifugatton and
resuspended m 20 mL of HEPES buffer and stored frozen at -70°C until required
(see Note 1) Lysis 1s performed by the addition of 250 Pg/mL lysozyme and
mcubatton on ice for 20 mm Cell debris 1s removed by centrtfugatron at 18,OOOg
for 20 mm and the resulting supernatant 1s the crude extract (see Notes 24)
2 Using lOO-pL altquots of crude supernatant the optimum percentage of
(NH&SO4 required to prectpttate Rl and R2 should be determined. Increasing
amounts of saturated solutton of the salt are added to gave a concentratton range
of between 2&50% saturation After incubation on ice for 20 min precrpitated
proteins are obtained by centrtfugatton at 13,000g for 20 mm and are resus-
pended m 100 ,ttL of HEPES buffer Supernatant and precrpitated fractions are
analyzed by SDS-PAGE and the lowest concentratton of (NH&SO, that gives
maximum prectpttatton of Rl and R2 determined For both proteins this is 35%
and m large-scale purtficattons finely ground (NH&SO4 powder gradually is
added to the crude extract on ice wtth constant sttrrmg After mcubatron on ice
for 20 min precipitated protems are obtained by centrifugatton at 12,OOOg for 20
mm and resuspended m a minimal volume of buffer (see Note 5)
3 The tsoelectrtc point of R2 should be determined using a Mono P chromato-
focusing column A pH range of 8.0-4 0 1s obtained using a 0 025Mtrtethanola-
mme/iminodtacettc acid buffer, pH 8.0 and Polybuffer 74/immunodtacettc actd,
pH 4 0 The R2 fraction (50 pL), partially purified by prectpltatton with 35%
(NH&SO4 should be diluted loo-fold and apphed to the Mono P column, prevt-
ously equthbrated with the pH 8 0 buffer. The flow-through fractton was col-
lected and bound protems were eluted by developmg a O-100% gradient of the
pH 4 0 buffer over 32 mL (see Note 6) The pH of each 1-mL fractton collected
was determined using a conventronal pH meter and elutlon of protein 1s analyzed
by SDS-PAGE. The column 1s washed with lMKC1 before reuse. The rsoelectrtc
point of R2 should be found to be pH 5.3.
4. Anion-exchange chromatography was performed on a 1-mL Mono Q column at
pH 5 8, 0 5 pH units above the pl of R2; at this pH R2 will bmd weakly to the
column and elute at low salt (see Note 7) The (NH&SO4 fraction is desalted
125
Protein Purification

1 2 3 4 5 6 7 6 910 11 12




Fig. 1. SDS polyacrylamide gel showing purification of R2 by Mono Q ion
exchange chromatography. Lane 1 shows the 35% (NH&SO4 fraction, lanes 2 and 3,
column flow-through material and lanes 4-12, proteins eluted from the column by the
(rlM KC1 gradient. R2 is indicated by an arrow.

using a Fast Desalt column into a 20 mA4 his-Tris-HC1 buffer pH 5.8 and applied to
the column (see Note 8). The column is then washed to remove unbound proteins
(flow-through fraction) and bound proteins are eluted with a O-l KC1 gradient
developed over I5 mL. Salt concentration is monitored by an in-line conductiv-
ity meter. Proteins present in each fraction are analyzed by SDS-PAGE (Fig. 1)
and R2 eluted as a distinct peak at 75 m&I KC1 (see Note 9). The salt gradient in
subsequent runs is adjusted to improve R2 resolution; following binding of pro-
teins to the column, the salt concentration was increased rapidly to 75 nnI4 KCl,
held at this for 5 column volumes, and then rapidly increased to 1M KCl. R2
produced by this method was greater than 95% pure (see Note 10). Although a
Mono Q column should be used in this purification similar results could be
obtained with a variety of other anion-exchange resins.
5. Chromatofocusing and ion exchange analysis of Rl indicated that these methods
are of limited use in Rl purification. Using the chromatofocusing protocol
described for R2, Rl elution in the pH 8.0-4.0 gradient should not be observed.
The behavior of Rl in anion and cation-exchange chromatography was analyzed
at a variety of pHs using buffers recommended for use with Mono Q and Mono S
columns. An FPLC Fast Desalt column should be used to alter the pH of the 35%
(NH&SO4 fraction to pH 4.0, 5.0,6.0, 7.0, 8.0, 9.0, and 10.0 and Mono Q and

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